DNA damage to the intestinal epithelium is associated with a number of pathological conditions, ranging from chemotherapy/radiation enteropathy to exposure to bacterial genotoxins and reactive oxygen species in the context of chronic inflammation/dysbiosis. Rapid and efficient epithelial regeneration is therefore critical for restoring barrier function and sequestering microbiota in the lumen. In the absence of injury, homeostatic turnover of the epithelium is maintained by a population of cycling intestinal stem cells (ISCs) at the crypt base. As these ISCs are highly sensitive to DNA damage-induced cell death, epithelial regeneration is driven by a DNA damage-resistant `reserve ISC' population. In the prior funding period, we demonstrated that activation of the Msi family of RNA binding proteins is both necessary and sufficient for cell cycle entry of reserve ISCs, and thus crucial for the regenerative response to DNA damage. However, the precise identity of this population has been a subject of contention, with recent findings suggesting that a host of lineage-committed epithelial cells (Paneth cells, transit-amplifying enterocyte progenitors, and secretory/enteroendocrine lineage cells (EECs) are capable of reverting to the ISC state once exposed to the niche environment. In our ongoing studies to characterize the reserve ISC, we generated a new mouse model harboring a CreERT2-2a-tdTomato cassette under control of the endogenous EEC-specific Chga locus (ChgaCreER2aTomato). Our preliminary data demonstrates that this allele faithfully captures cells across the EEC lineage, from immature progenitor to mature EEC. Further, lineage tracing from these cells verifies that a significant proportion of regeneration after DNA damaging injury is derived from the ChgaCreER2aTomato population, suggesting that this population is uniquely required for this process. Here, we test the hypothesis that EEC- lineage cells are required for regeneration after DNA damage and that this process is controlled by specific Msi-RNA interactions. Further, we hypothesize that cells of the EEC lineage reach an epigenetic `point of no return' after which their plasticity is lost. To address these hypotheses, we combine novel genetically modified mouse models with single cell genomic and functional assays, including an inducible Msi2-HyperTRIBE allele which enables the identification of direct Msi2 binding targets in rare EEC lineage cells in vivo, as well as histone H2B-GFP pulse-chase assays that enable us to assess how the latent stem cell potential of EEC lineage cells changes as a function of their age. Ultimately, the experiments in this proposal employ state-of- the art single cell genomic and functional approaches to gain insight into the molecular basis for epithelial regeneration from a rare but incredibly powerful cell population. Findings from this work will inform the development of targeted strategies to prophylactically guard against intestinal injury or to enhance the regenerative response post-injury.